1. Field of the Invention
The invention generally relates to controlling the activity of a protein on a substrate.
2. Description of the Related Art
ATPases such as kinesin are central to several life processes. These proteins are found in organisms representing all of the major eukaryotic kingdoms. The common bond among the kinesins is a highly conserved “motor” domain, ˜350 amino acids long, which contains binding sites for ATP and cytoskeletal microtubules. Kinesins are intimately associated with the microtubules, and in concert with them drive cell division processes, mediate intracellular transport of organelles, chromosomes, and RNA. In vivo, kinesin activity is moderated reversibly and irreversibly via enzymatic phosphorylation of specific amino acid residues and chemical inhibitors.
Kinesin is allosteric, that is, a ligand binding event in a given region of the protein can cause a conformation change in a distant region. If the conformationally-affected region contains the catalytic site, the enzyme may exhibit an increased or decreased ability to bind and/or transform substrate. (Marvin et al., “The rational design of allosteric interactions in a monomeric protein and its applications to the construction of biosensors”, Proc. Natl. Acad. Sci. USA, 94, 4366(1997). All referenced publications and patent documents are incorporated herein by reference.) In the literature there is one example of an allosteric “switch” artificially introduced into F1-ATPase (closely related to kinesin) via protein engineering. In this example, the “switch” is a pocket that binds a Zn++ ion. When the ion is bound, the ATPase loses roughly 75% of its activity. The “switch” is, however, not reversible. When the ion is removed via chelation, the ATPase activity is partially, but not fully, restored (Liu et al., “Control of a biomolecular motor-powered nanodevice with an engineered chemical switch”, Nature Mat., 1, 173 (2002)). The catalytic activity of redox enzymes (enzymes that use electrons as a substrate) has been controlled using conducting polymer surfaces (Ryder et al., “Role of conducting polymeric interfaces in promoting biological electron transfer”, Biosensors & Bioelectronics, 12, 721 (1997); Liu et al., “Enzymatic activity of glucose oxidase covalently wired via viologen to electrically conductive polypyrrole films”, Biosensors & Bioelectronics, 19(8), 823-834 (2004)). In these cases, the polymer merely served as a conduit for the electron substrate, with higher currents allowing an increased enzyme activity because of the increased number of electrons available. The catalytic activity of urease (not a redox enzyme) reportedly has been controlled using a conducting polymer surface. In this case, an electron is able to reduce a disulfide bond in the active site, generate a catalytic thiol, and thus activate the enzyme (Uchiyama et al., “Electrical control of urease activity immobilized to the conducting polymer on the carbon felt electrode”, Electroanalysis, 14, 1644 (2002)).
Protein structural changes can also be caused by the charged surfaces of minerals such as montmorillonite and other clays. For example, bovine serum albumin and chymotrypsin undergo significant changes in folding/conformation (shown by FTIR), and catalytic activity as they adsorb to these inorganic surfaces.
There have been disclosures concerning the combination of conducting polymers/surfaces and proteins describing the conducting surface as a conduit for information transduction (such as detection of a binding event), or for electron supply for redox proteins (Koopal et al., U.S. Pat. No. 5,422,246; Guiseppe-Elie, U.S. Pat. No. 5,766,934; Albarella et al., U.S. Pat. No. 5,210,217; Charych et al., U.S. Pat. No. 6,660,484).